Immunohistochemical staining was performed on 5–μm sections of formalin-fixed, paraffin-embedded tissue using antibody to Vimentin (NCL-L-VIM-V9; Novocasrtra, UK) and E-Cadherin (NCH-38; Dako, CA USA). The visualization system used was BenchMark XT with heat-induced epitope retrieval (CC1 solution, Ventana) and iView DAB detection kit (Ventana, Tucson, AZ).
Nch 38
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The NCH-38 is a laboratory instrument designed for electrochemical analysis. It is capable of performing a range of electrochemical measurements, including cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy. The core function of the NCH-38 is to provide researchers and scientists with a versatile tool for investigating and characterizing electrochemical systems.
Automatically generated - may contain errors
Lab products found in correlation
Cc1 solution, by Roche (2 mentions)
Benchmark xt, by Roche (2 mentions)
Iview dab detection kit, by Roche (2 mentions)
Envision flex, by Agilent Technologies (2 mentions)
Dako envision kit, by Agilent Technologies (1 mentions)
Liquid permanent red, by Zytomed Systems (1 mentions)
Clone 6g11, by Agilent Technologies (1 mentions)
Prestige antibody, by Merck Group (1 mentions)
Real envision detection system, by Agilent Technologies (1 mentions)
Ep700y, by Cell Marque (1 mentions)
Ultraview universal dab detection kit, by Roche (1 mentions)
E cadherin, by Agilent Technologies (1 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Dp21 digital camera, by Olympus (1 mentions)
β catenin, by Leica (1 mentions)
Vimentin v9, by Leica (1 mentions)
Cx41 microscope, by Olympus (1 mentions)
Envision hrp kit, by Agilent Technologies (1 mentions)
Ac 15, by Merck Group (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
11 protocols using nch 38
1
Immunohistochemical Evaluation of Vimentin and E-Cadherin
2
Quantitative EGFR and E-Cadherin Analysis
3
Evaluating Tumor Infiltration and Adhesion
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Tissue microarrays with a core size of 1 mm respectively 1.5 mm in diameter (two cores per sample) were performed of FFPE resection specimens. Then standard hematoxylin/eosin (H&E) staining as well as immunohistochemistry was performed. The following primary antibodies were used: anti-CD15 (DAKO, IS062, 1:50, retrieval condition: pH 6.0), anti-E-cadherin (DAKO, NCH-38, 1:10, retrieval condition: pH 9.0), ZEB1/AREB6 (abcam, 416A7H10, 1:200, retrieval condition: pH 6.0) As secondary antibody either the Histofine, Simple Stain universal polymer was used (Nichirei, Tokyo, Japan), followed by the color reaction using liquid permanent red (Zytomed, Berlin, Germany), or the Dako EnVision kit (Dako) was used, both followed by counter stain with hematoxylin. The amount of E-cadherin expression was determined using the Allred score (score 0 - 8), calculated by the sum of staining intensity (0= no; 1= weak; 2= moderate; 3= strong) and distribution (0= 0%; 1= <1%; 2= 1-10%; 3= >10-33%, 4= >33-66%, 5= >66-100% positive cells). The number of tumor infiltrating neutrophils was judged by a semiquantitative scoring system (negative: score 0 = 0, moderate: score 1 =1-19, high: score 2 = ≥ 20 PMN/ 1 mm diameter core) and the nuclear translocation of ZEB1 was judged present or absent (0 = absent; 1= present).
4
Immunohistochemical Assessment of Wnt5a, N-Cadherin, and E-Cadherin in Cancer Cells
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In the immunohistochemistry cohort, IHC was performed with mouse monoclonal antibodies against Wnt5a (Sigma-Aldrich, clone 3A4, dilution 1:50), N-cadherin (Dako, clone 6G11, dilution 1:30), and E-cadherin/NCH-38 (Dako, clone NCH-38, dilution 1:100) and polyclonal rabbit antibodies against β-catenin/CTNNB1 (PRESTIGE antibodies Sigma, dilution 1:300). The sections were counter-stained with Haematoxylin. Assessment was performed manually, and all the IHC sections were evaluated based on the average staining intensity (0-3) multiplied by the percentage of positive cancer cells (0-3), obtaining a total staining index (SI) (0-9). A SI of 0 was regarded as negative, 1-2 as weak positive staining; 3-6 as moderate, and 9 as strong positive staining (Supplementary Table 7 ). An experienced pathologist (AMB) validated the scoring.
5
Evaluating Epithelial-Mesenchymal Transition Markers in PSRCC and MBC
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In order to identify the biological background that can result in the difference between PSRCC and MBC, we evaluated the status of Vimentin, and E-cadherin using immunohistochemical analysis. Primary antibody against Vimentin (790–2917, Roche, Ventana) and E-cadherin (NCH-38, DAKO, Denmark) was detected using the DAKO REAL EnVision Detection System. Immunostaining intensity of Vimentin and E-cadherin was estimated in three categories: 0 (0–10%), 1+ (11–25%), 2+ (26–50%), and 3+ (>51%), and a cutoff value of>25% was defined as over-expression.
6
E-cadherin IHC Protocol
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E-cadherin IHC was performed using either clone EP700Y (Cell Marque, dilution 1 : 200) and the Ventana Benchmark autostainer or clone NCH-38 (DAKO; Cat. #M3612, dilution 1 : 50) followed by the UltraView Universal DAB Detection Kit (Ventana Medical Systems).
Any membranous immunoreactivity, irrespective of the number of cells or the intensity, was scored as positive. The absence of membranous immunoreactivity or the presence of cytoplasmic stain only was scored as negative.
Any membranous immunoreactivity, irrespective of the number of cells or the intensity, was scored as positive. The absence of membranous immunoreactivity or the presence of cytoplasmic stain only was scored as negative.
7
Immunohistochemical Analysis of Tissue Markers
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Tissue sections of 5 μm were tested against E-cadherin (NCH-38, prediluted format, Dako), β-catenin (17C2, prediluted format, Novocastra), vimentin (V9, 1/500 Novocastra), NOS2 (SAB4502012, 1/1000, Sigma), NF-κB (p65, 2A12A7, 1/500, Invitrogen), TGF-β (TB21, 1/1000, Thermofisher), IL-6 (10C12, 1/100, Novocastra), IL-17 (50104, 1/100, Invitrogen), IL-10 (945A2A5, 1/100, Invitrogen), CD68 (KP1, prediluted format, Dako), and MMP-9 (2C3, 1/200, Santa Cruz Biotechnology). Formalin-fixed, paraffin-embedded sections were deparaffinized, hydrated, and treated using a high temperature for antigen retrieval in EnVision™ FLEX Solution (Dako). After blocking of endogenous peroxidase, sections were incubated 1h at 37°C with appropriate primary antibodies. Immunorevelation and counterstaining were done with EnVision™ FLEX (Dako). The sections were observed and photographed using an Olympus microscope CX41 equipped with a DP21 Olympus digital camera.
8
Immunohistochemistry of SQLE and E-cadherin in CRC
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For the IHC, all cases of CRC tissue with accompanying normal epithelium were fixed in 10% buffered formalin for 24 to 48 h and embedded in paraffin. Immunostaining was performed on tissue sections of 4 µm thickness, using IgG-rabbit polyclonal antibody against SQLE (1:100 dilution; Interchim, Montluçon, France) and mouse monoclonal antibody against E-cadherin (NCH-38, 1:100 dilution; DakoCytomation, Glostrup, Denmark) as primary antibodies. Paraffin-embedded tissue sections were deparaffinized and rehydrated through a series of xylene and graded ethanol, and autoclaved at 120 °C for 10 min with 10 mM/L sodium citrate buffer (pH 6.0). IHC conditions for SE and E-cadherin were optimized according to the manufacturers’ instructions. The slide sections were incubated with the primary antibodies for 90 min and stained with 3,3′-diaminobenzidine as the substrate using an EnVision-HRP kit (DakoCytomation, Glostrup, Denmark). Negative controls were obtained by using an irrelevant mouse IgG of the same isotype. All slides were counterstained for 1 min using Mayer’s hematoxylin and then mounted.
9
Epithelial-Mesenchymal Transition Markers
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Cells were exposed to E2hi and P or vehicle in TGFβR2i without EGF for 10 days, depleted of hormones for 7 days, split and cultured in TGFβR2i without EGF and 2i for 4 days before extraction and blotting as described5 using antibodies against E-cadherin (1:1000, NCH-38, DAKO), vimentin (1:1000, V9, DAKO), c-KIT (1:1000, clone 47233, R&D Systems), p27 KIP1 (1:1000, clone 57, BD Biosciences), and β-actin (1:5000, AC-15, Sigma). That markers were regulated irrespective of media condition was confirmed in an alternative set-up with E2hi and P or vehicle exposure for 13 days in TGFβR2i-1 with CCS, depletion for 14 days, split and exposure to TGFβR2i-1 for 3 days prior to Western.
10
Immunohistochemical Profiling of Tissue Samples
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Immunohistochemical staining was performed on 5-μm sections of formalin-xed, para n-embedded tissue using antibody to Vimentin (NCL-L-VIM-V9; Novocasrtra, UK) and E-Cadherin (NCH-38; Dako, CA USA). The visualization system used was BenchMark XT with heat-induced epitope retrieval (CC1 solution, Ventana) and iView DAB detection kit (Ventana, Tucson, AZ).
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